PROJECT 2 PROJECT SUMMARY Cystic fibrosis (CF) is caused by loss-of-function mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) apical membrane anion channel. Loss of CFTR- mediated HCO3- and Cl- secretion by airway epithelia impairs respiratory host defenses, causing bacterial infection, inflammation, mucus accumulation, and respiratory failure. One strategy for restoring anion channel function to the apical membrane is to increase the function of mutant CFTR channels. That has proven successful for people with specific CFTR mutations. However, ~10% of people with CF have mutations that do not respond to CFTR modulators or they are not able to take modulators. Another strategy is to provide an alternative channel. Toward this end, we studied amphotericin B (AmB); earlier work showed that AmB forms anion channels. We discovered that apical AmB increased HCO3- and Cl- secretion in cultured CF airway epithelia, including those with CFTR-null mutations. AmB increased ASL pH, height, and antibacterial activity. AmB is clinically approved to treat fungal infections, and off-label lung aerosolization has an impressive safety record. Thus, AmB could potentially provide a new mutation-agnostic therapy for CF lung disease. However, many questions remain unanswered and we lack important in vivo data. Therefore, our overarching goal is to understand the mechanisms of AmB-induced anion secretion in airway epithelia and to test the hypothesis that AmB can restore CF host defenses in vivo. To achieve this goal, we will answer questions in three aims. Aim 1. What molecular and cellular mechanisms elicit AmB-mediated anion secretion? Without cAMP-dependent regulation and with half the anion selectivity of CFTR, AmB is an imperfect substitute for CFTR. We will test key hypotheses about how AmB functions in airway epithelia. Aim 2. Which epithelial cells does AmB target and how does AmB alter them? These studies will reveal AmB function in large and small airway epithelia and diverse cell-types, test AmB in airway epithelia remodeled by inflammation, and test for compensatory changes. Aim 3. Does AmB reverse CF host defense defects in vivo? Using CF pigs, we will test if nebulized AmB increases ASL pH, increases ASL antimicrobial activity and height, increases mucociliary transport, and enhances S. aureus eradication from the lungs. The results of these studies will have direct implications for developing a new genotype agnostic approach to treating CF, and will inform development of other therapies in Projects 1 and 3. Our proposal?s success is enhanced by an ongoing collaboration between the labs of Welsh (Iowa) and Burke (Illinois), by utilization of outstanding cores, and by an environment of cooperation between the Program?s projects and investigators. With this background, our track record, and our commitment, we believe this research can change the lives of people with CF.